Multi-arm histidine copolymer for controlled release of insulin from poly(lactide-co-glycolide) microsphere Wooram Park a, b , Dongin Kim b , Han Chang Kang c , You Han Bae b, d, ** , Kun Na a, * a Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea b Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, 421 Wakara Way, Suite 318, Salt Lake City, UT 84108, USA c Department of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea d Utah-Inha Drug Delivery Systems (DDS) and Advanced Therapeutics Research Center, 7-50 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea article info Article history: Received 3 August 2012 Accepted 17 August 2012 Available online 6 September 2012 Keywords: Insulin Multi-interaction complex Polyelectrolyte complex PLGA microsphere Protein delivery Sustained protein delivery abstract For long-term, sustained protein delivery, a new, star-shaped block copolymer composed of methoxy poly(ethylene glycol) (mPEG), branched oligoethylenimine (bOEI), and poly(L-histidine) (pHis) was synthesized via the multi-initiation and ring-opening polymerization (ROP) of His N-carboxy anhydride (NCA) on bOEI with a PEG conjugation. The resulting mPEG-bOEI-pHis (POH) had strong buffering capacity within the neutral-to-acidic pH range and was complexed with insulin (Ins) via an electrostatic attraction plus hydrophobic interactions, resulting in the formation of a dual-interaction complex (DIC, weight ratio 2) of approximately 30e60 nm in size. This DIC tolerated high salt concentrations without destabilization, supporting the existence of hydrophobic interactions, and protected Ins from the organic solvent/water interface. The DIC in poly(lactide-co-glycolide) microspheres (PLGA MS) as a long-term Ins delivery formulation was evenly distributed via a double-emulsion method. The DIC-loaded PLGA MS offered a higher Ins loading and a lower initial burst than Ins-loaded PLGA MS. This formulation possessed near zero-order release kinetics (for at least one month). In streptozotocin (STZ)-induced diabetic rats, a DIC-loaded PLGA MS formulation was able to maintain blood-glucose levels at 200 e350 mg/dL for the first two weeks and even lower levels (100e200 mg/dL) for the next two weeks. Thus, a new POH polymer and its complex with a drug protein could have potential biological application as a long-term, sustained protein delivery system. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Poly(lactide-co-glycolide) (PLGA) microspheres (MS) have been intensively investigated as storage systems for long-term protein delivery [1e3]. However, proteins encapsulated in PLGA MS are often exposed to acidic environments that lead to unwanted hydrolytic PLGA byproducts (e.g., glycolic acid and lactic acid) [4]. As a result, the cargo proteins can become denatured, leading to their reduced bioactivity [5,6]. To prevent or reduce acid-induced complications, the use of various polymeric additives has been investigated [7e13]. In one method, a polyelectrolyte complex (PEC) was formed via ionic interactions between charged proteins and counter-charged poly- mers [10e12]. The PEC physically isolated the proteins from the PLGA matrix, resulting in improved protein stability. However, the polymer/protein complex dissociated during the fabrication of the PLGA MS and readily released proteins under physiological condi- tions due to weak ionic interactions [10]. To reduce the rate of release, chemical crosslinking was introduced into the core or shell of the PEC [14]. The crosslinked PEC, however, still exhibited poor biocompatibility and biodegradability and caused unwanted chemical crosslinking between proteins and polymers within and between the PECs (i.e., inter-PEC crosslinking) [15]. Another method applied polyelectrolytes (e.g., poly(L-histidine) (pHis) and oligo(vinylsulfadimethoxine) (OVSDM)) that have a proton buffering capacity in acidic pH [9,13,16,17]. These poly- electrolyte/protein complexes significantly enhanced protein stability and release kinetics but still exhibited weak binding affinity at neutral pH [9,13]. This method demanded a stronger binding interaction with the protein and better buffering against * Corresponding author. Tel.: þ82 2 2164 4832; fax: þ82 2 2164 4865. ** Corresponding author. Department of Pharmaceutics and Pharmaceutical Chemistry, The University of Utah, 421 Wakara way, Suite 318, Salt Lake City, UT 84108, USA. Fax: þ1 801 585 3614. E-mail addresses: you.bae@utah.edu (Y.H. Bae), kna6997@catholic.ac.kr (K. Na). Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.08.042 Biomaterials 33 (2012) 8848e8857